Artículo original Alvarado-Delgado A y col.

Ecdysis-related pleiotropic expression during albimanus development

Alejandro Alvarado-Delgado, MSc,(1) Ken Moran-Francia, Biol,(1) Guillermo Perales-Ortiz, MSc,(1) Mario Henry Rodríguez, PhD,(1) Humberto Lanz-Mendoza, PhD.(1)

Alvarado-Delgado A, Moran-Francia K, Alvarado-Delgado A, Moran-Francia K, Perales-Ortiz G, Rodríguez MH, Lanz-Mendoza H. Perales-Ortiz G, Rodríguez MH, Lanz-Mendoza H. Ecdysis-related pleiotropic Expresión de neuropéptidos pleiotrópicos neuropeptides expression during asociados con ecdisis durante el desarrollo Anopheles albimanus development. del Anopheles albimanus. Salud Publica Mex 2018;60:48-55. Salud Publica Mex 2018;60:48-55. http://doi.org/10.21149/8134 http://doi.org/10.21149/8134

Abstract Resumen Objective. To analyze the transcription pattern of neu- Objetivo. Describir la expresión de neuropéptidos durante ropeptides in the ontogeny of a malaria vector, the mos- la ontogenia del mosquito vector de la malaria Anopheles quito Anopheles albimanus. Materials and methods. The albimanus. Material y métodos. Se midió la expresión de transcription pattern of Crustacean CardioActive peptide CCAP, corazonina, ETH, allatostatina, orcokinina, ILP2, ILP5 y (CCAP), corazonin, Ecdysis Triggering (ETH), bursicon en de primer (2mm), segundo (4mm), tercer allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin (5mm) y cuarto (6mm) estadio, y adultos, Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR mediante qPCR. Resultados. A diferencia de otros insectos on larvae (1st – 4th instar), pupae and adult mosquitoes. Re- en donde, CCAP, corazonina y ETH se expresan principal- sults. Unlike in other , transcripts of CCAP (70.8%), mente en estadios pupales, en An. albimanus se expresaron ETH (60.2%) and corazonin (76.5%) were expressed in 4th mayoritariamente en larvas de cuarto estadio, CCAP tuvo instar larvae, probably because these three neuropeptides are 70.8% de expresión relativa, corazonina 76.5% y ETH 60.2%. associated with the beginning of ecdysis. The ILP2 fue el neuropéptido que más se expresó en el primer, ILP2 showed higher transcription levels in other stages and segundo y tercer estadio y orcokinina disminuyó durante el orcokinin decreased during the development of the mosquito. desarrollo del mosquito. Conclusión. Los péptidos estu- Conclusion. The CCAP, corazonin and ETH neuropeptides diados se expresaron en todos los estadios de desarrollo del are potential targets for the design of control strategies aimed mosquito. Sin embargo, su expresión varió en cada uno de at disrupting An. albiamnus larval development. ellos. Los neuropéptidos CCAP, corazonina y ETH, que son esenciales para la transformación de lavas a pupas, pueden ser blancos potenciales para el diseño de estrategias de control dirigidas a interrumpir el desarrollo larvario de An. albimanus. Keywords: gene expression; neuropeptides; Anopheles; Palabras clave: expresión génica; neuropéptidos; Anopheles; insecto

(1) Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública. Cuernavaca, Morelos, México.

Received on: August 18, 2016 • Accepted on: February 24, 2017 Corresponding author: Dr. Humberto Lanz-Mendoza. Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública. Av. Universidad 655 col. Santa María Ahuacatitlán. 62100 Cuernavaca, Morelos, México. E-mail: [email protected]

48 salud pública de méxico / vol. 60, no. 1, enero-febrero de 2018 Anopheles albimanus neuropeptides during development Artículo original

nsects play prominent roles in supporting human Understanding the expression of these neuropep- welfare (as food sources and crop pollinators), a tides in developmental stages of insect vectors may numberI of hematophagous transmit several open opportunities for identifying possible candidate infectious diseases.1 Malaria and various viral diseases molecules to design species-specific insecticides for transmitted by Anopheles, Aedes, and Culex mosquitoes2 biological control.9 In this paper, we examined the affect about 1 000 million people and produce about expression of eight neuropeptides, three associated to one million deaths worldwide yearly. No effective vac- ecdysis, during the ontogeny of the mosquito Anopheles cines are available and the main control strategies are albimanus, one of the main malaria vectors in Mexico directed to abate mosquito vector populations and to and Central America.31 prevent human-vector contact.3-5 Chemical insecticides are the main weapons used to control these vectors, but Materials and methods in insecticide-resistance is an important limitation. In recent decades, the interest in bio-insecticides target- Biological samples of Anopheles albimanus ing molecules that disrupt mosquito development,6 has increased.7 Larval, pupae and adults mosquitoes were obtained from Neuropeptides have pleiotropic functions in the insectary of the Center for Research on Infectious insects’ physiological processes, including reproduc- Diseases (CISEI), National Institute of Public Health tion, development, metabolism, and behavior.8 These (Mexico). Larvae were grown under controlled tempera- could be specific targets for vector control with little ture conditions (29-33°C) and humidity (70-80%), with a environmental hazards.9 Of particular interest are those 12h light/12h dark photoperiod. Larvae were fed with engaged in mosquito and ecdysis, such grinded cat chow (Whiskas 0.1876 grams every 24 hours) as: Crustacean CardioActive Peptide (CCAP), Ecdysis in trays with 1.5 liters of water at 25-30°C at larval density Triggering Hormone (ETH), corazonin and bursicon. of 200 each tray. Larval samples were taken every 24 h CCAP is a cyclic nonapeptide that stimulates heart- after egg hatching and adult mosquitoes were collected beat10 after adult emergence,11 oviduct contraction12 24 hours after emergence. Because it was difficult to dif- and plays an essential role in ecdysis and eclosion.13 ferentiate between larvae instars by morphology, larvae Corazonin is a peptide, whose functions are associated were measured and defined as first instar, larvae of 2 mm, with cardioacceleratory activity,14 diapause, circadian second instar, larvae of 4 mm, third instar, larvae of 5mm rhythm and induction of melanization,15 and bursicon, and fourth instar, larvae of 6 mm in length. Groups of 8 a heterodimer required for sclerotization, cuticle tanning larvae, 8 pupae and 8 adult mosquitoes were placed in and wing expansion after eclosion.16 Prior to ecdysis, 500 μl of Trizol (Ambion) and stored at -20°C until use. corazonin is released into the haemocel and activates the release of pre-ecdysis triggering hormone (PETH) Total RNA purification and ETH.17 ETH is a peptide that activates neurons that produce the eclosion hormone (EH), CCAP and Total RNA was extracted as previously described.32 bursicon.18 Other important neuropeptides involved in Briefly, samples in Trizol were macerated with a biovo- growth and development are the insulin like peptides rtex, 10 pulses per 1 minute with a 30 second break. (ILP´s), orcokinins and allatostatins. ILP´s seem to be Samples were centrifuged for 5 min at 8 000 x g to re- involved in development, regulation of carbohydrate move the debris, the supernatant was added to 100 µl of and lipid metabolism,19 diapause induction,20 stress chloroform (Sigma-aldrich), mixed and centrifuged for resistance,21 regulation of sexual behaviors,22 regulation 15 min at 10 000 x g at 4°C, the aqueous phase was re- of life span23 and regulation of sleep.24 In Aedes aegypti, covered and 250 µl of cold isopropanol (Sigma-Aldrich) insulin was shown to stimulate ecdysteroid biosynthesis were added, mixed and incubated at -20 °C for 1 hour. in the ovaries.25 Allatostatins, are potential insect growth Samples were centrifuged at 10 000 x g for 10 min, the regulators, which inhibit the production of juvenile pellets were washed with 500 µl of 75% ethanol and hormone (JH);26 although it appears to have other func- centrifuged at 7 000 x g for 5 min; the supernatants tions, including neuromodulation, regulation of muscle were removed and the pellets were-suspended in 20 µl contraction, and regulation of enzyme biosynthesis.27 of DEPC-treated water (diethylpyrocarbonate) (Sigma- Orcokinins are involved in the neuronal regulation of aldrich). RNA was quantified with Nanodrop and ecdysteroidogenesis28 and circadian locomotor activity29 visualized using electrophoresis in agarose gels stained and the control of vitellogenesis.30 with ethidium bromide, to confirm integrity.

salud pública de méxico / vol. 60, no. 1, enero-febrero de 2018 49 Artículo original Alvarado-Delgado A y col. cDNA Synthesis Neuropeptide expression associated to Ecdysis One µg of total RNA of each sample was treated with DNAse (ThermoScientific® ™ 200 U/µl). cDNA was The transcription pattern of CCAP, corazonin and synthetized, the reaction included: 250 ng oligodT ETH transcripts varied among the different mosquito (Thermo Scientific), 5X RT buffer (Promega), 2.5 mM developmental stages. The highest expression of the dideoxynucleotide mixture (dNTP’s) (Promega), 10U/ three neuropeptide transcripts was observed in fourth µl RNAse Inhibitor (RNAsin) and 5 U/µl reverse tran- instar larvae (70.8, 76.5 and 60.2%, respectively) (figure scriptase enzyme (Promega). All samples were adjusted 1). CCAP transcription increased from first to third to a final volume of 40 µl of DEPC-treated water and instar larvae (1.81 to 21.3%) but, in pupae and adult incubated at 42 °C for 90 minutes. The synthesized mosquitoes, transcription levels decreased to 4 and cDNAs were stored at -70 °C until use. 0.3%, respectively (p< 0.001) (figure 1A). The corazo- nin transcript had similar expression profile, in first Quantitative real time PCR (qPCR) to third instar larvae, transcription levels were 4 to 1.6%, respectively but, in pupae and adult mosquitoes, Specific oligonucleotides corresponding to CCAP, transcription levels decreased to 0.1 and 2.5%, respec- corazonin, ETH, ILP2, ILP5, allatostatin-A, orcokinin, tively ( < 0.001) (figure 1B). The transcription of ETH and bursicon genes were designed using the Oligo 3.1 increased from first to third instar larvae (0.1 to 13.7%, Program Analyzer (http://www.idtdna.com/ site), respectively), in pupae the transcription decreased to based on the nucleotide sequences identified in the 1.5% but in adult mosquitoes, it increased to 14.5% genome (https://www.vectorbase.org/organisms/ (p< 0.001) (figure 1C). A schematic representation of anopheles-albimanus) and a brain transcriptome of the neuropeptide variation among mosquito stages is An. albimanus.33 All quantitative RT-PCR assays were shown in figure 1D. performed in duplicate with three biological replicates for each experiment. As an endogenous control, an ILP 2, ILP5, bursicon, allatostatin-A oligonucleotide corresponding to the actin gene of and orcokinin An. albimanus was used. Real-time PCR assays were performed on an Applied Biosystems (ABI) Step One The transcription of ILP 2 was a high in most stages: 47% Plus Real-Time PCR System using the reaction mixture (first instar), 45.5% (second instar), 45% (third instar), Master Mix (2X) SYBRGreen universal qPCR (Thermo- 64.5% (pupae) and 61.6% (mosquito) (p< 0.001), the low- Scientific). The PCR program was: 95 °C for 10 min, est transcription level of ILP2 was in fourth instar larvae followed by 40 cycles of 95 °C for 15 seconds and 64 °C (1.2%) (figure 2). Allatostatin-A and bursicon transcrip- for 1 min, then 95 °C for 15 sec, 64 °C for 15 sec, and tion occurred in fourth instar larvae. ILP5 expression 95 °C for 15 sec, for one cycle. The specificity of the decreased as the mosquito development progressed: 9, SYBR green PCR signal was confirmed by a melting 6.5, 1.4, 0.2 and 0.1% in first, second, third and fourth curve analysis and 1.2% agarose gel electrophoresis. instar larvae and pupae, respectively; but increased again The expression of each neuropeptide in different in adult mosquitoes (6.5%) to similar values to that of mosquito development stages was compared using fourth instar larvae (p< 0.001) (figure 2). The transcription the 2-DDCt method34 and calculated as fold increase of bursicon increased from first instar (11.2%) to third comparing the expression of each neuropeptide in each instar larvae (34.2%). Its transcription decreased in fourth stage against actin and tested with one-way ANOVA instar larvae (1.5%), increased again in pupae (27.6%), but followed by the Kruskal-Wallis post-test (α= 0.05). in adult mosquitoes the transcript expression was lower Finally, graphical data were shown as percentage of (18.3%) (p< 0.001) (figure 2). No detectable transcription expression by stage. variations during the mosquito development were ob- served for orcokinin and allatostatin-A. The transcription Results pattern of orcokinin was 15.5, 0.7, 5.3, 2, 0.4, and 0.1% in first, second, third and fourth instar larvae, pupae and The transcription pattern of CCAP, corazonin, ETH, adult mosquitoes, respectively. The transcription pattern ILP2, ILP5, bursicon, allatostatin-A and orcokinin neu- of allatostatin-A was 2.3, 4.94, 2.9, 1.1, 4.2, and 5.8% in ropeptides in larvae from first to fourth instar, first, second, third and fourth instar larvae, pupae and black and mosquito was measured. adult mosquitoes, respectively (figure 2).

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A B 80 CCAP 100 Corazonin

80 60

ession ession 60 40 e expr e expr 40

20

% Relativ % Relativ 20

0 0 First Second Third Fourth Pupae Adult First Second Third Fourth Pupae Adult instar instar instar instar instar instar instar instar

C D 100 ETH

80

ession 60 e expr 40

% Relativ 20 1st 2nd 3rd 4th Pupae Adult 0 Instar First Second Third Fourth Pupae Adult instar instar instar instar Cor ETH CCAP

CCAP: Crustacean Cardio Active Peptide ETH: Ecdysis Triggering Hormone qPCR: Quantitative polymerase chain reaction

Figure 1. Variation of expression levels of CCAP, corazonin and ETH in different developmental stages of Anopheles albimanus measured by qPCR. The three neuropeptides had more 60% of expres- sion. A) CCAP (70.8%), B) corazonin (76.5%) and C) ETH (60.2%). D) In fourth instar larvae

Discussion ity and bioavailability have been studied for their use in pest control. For example, a β-amino acid pyrokinin In this paper we analyzed the transcription pattern analog (Ac-Y[β3F]TPRLamide) accelerates an irregular of eight neuropeptides, during the development of pupation in the flesh Sarcophaga bullata.37 A PK/ An. albimanus mosquitoes. Mosquitoes have a well- PBAN antagonist lead (RYF[dF]PRLa) was reported characterized development process from egg hatching as a potent inhibitor of a sex pheromone biosynthe- to adults. Neuropeptides are essential during their sis in Heliothis peltigera.38 Using a diapause hormone ontogeny and together with their receptors, have been (DH) agonistic and/or antagonistic peptidomimetics proposed as candidates for the generation of larvicides the pupal diapause of Helicoverpa zea was disrupted39 that disrupt their development. Currently, a number of and analogs of the pyrokinin, insect kinin and insect 35 peptidomimetics with c-terminal motifs PRXamide tachykinin families showed aphicidal activity, that was 36 and FGLamide that confer an increase in their stabil- better than some commercially available aphicides.40

salud pública de méxico / vol. 60, no. 1, enero-febrero de 2018 51 Artículo original Alvarado-Delgado A y col.

ILP 2 ILP 5 80 25 60 20 40 15 10

ession 20 ession 5 3 3 e expr e expr

2 2

% Relativ 1 % Relativ 1

0 0 First Second Third Fourth Pupae Adult First Second Third Fourth Pupae Adult instar instar instar instar instar instar instar instar

Bursicon Orcokinin 50 30 40 30 20 20

ession ession 10 10 5 3 e expr e expr 4 3 2

% Relativ 2 % Relativ 1 1 0 0 First Second Third Fourth Pupae Adult First Second Third Fourth Pupae Adult instar instar instar instar instar instar instar instar

Allatostatin-A 20 15 10 ession 5 3 e expr

2

% Relativ 1

0 First Second Third Fourth Pupae Adult instar instar instar instar

ILP: insuline like peptides

Figure 2. Variation of expression levels of orcokinin, ILP5, ILP2, allatostatin and bursicon in different developmental stages of Anopheles albimanus measured by qPCR. ILP2 showed higher ex- pression in different stages (first, second, third and fourth larvae instar, pupae and mosquitoes)

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In mosquitos , only CCAP,3 FMRFamide-like peptides emergence adult stages, it is clear that many of the (FLP),41 corazonin,42 bursicon,43 short neuropeptide F neuropeptides are expressed in different intensities (sNPF),44 AKHI and AKHII45 have been studied during or are specific to different stages; and it would be the ontogeny of An. gambiae. interesting to also investigate their expression in eggs This is first report of the expression of neuropep- and post emergence individuals. We also recognize tides associated to several functions during ontogeny that other important stages as eggs, brown pupae and of An. albimanus. The high transcription of CCAP, adult stages older than 24 hours post emergency re- ETH and corazonin in fourth instar larvae suggests quire further studies. However, the encouraging results that these neuropeptides associated with ecdysis be- of the present study will hopefully stimulate further gin their expression in this instar, and that corazonin studies that will explore neuropeptides as target mol- and CCAP are required to activate ETH and start the ecules in larval control of diseases-transmitting insects. ecdysis. Unlike these results, CCAP and corazonin are Undoubtedly detailed studies are needed to identify expressed in different developmental stages of An. specific candidates to fight anophelines mosquitoes, gambiae,31 Specifically, CCAP mRNA levels increased vectors of malaria, a disease that remains a public in second instar larvae, decreased in third instar larvae health problem worldwide. and then increase to maximum levels in callow pupa stage.3 While corazonin showed a similar transcription Acknowledgments profile in larvae of An. gambiae and An. albimanus the increase to maximum levels in An. gambiae occurred This study was supported by the Consejo Nacional in young adults.42 We speculate that the different de Ciencia y Tecnología de México (Conacyt) (Grant expression pattern of CCAP, corazonin and ETH dur- 243171). Alejandro Alvarado Delgado is a PhD stu- ing the ontogeny of An. albimanus and An. gambiae is dent in Programa de Doctorado en Ciencias Biomédicas due to factors associated to development, such as: UNAM, and appreciates the support given by Conacyt temperature, food and stress; but also to different (Beca 98230). environmental adaptations of both mosquito species Declaration of conflict of interests. The authors declare that they have no such as resistance to insecticides, colonization of areas conflict of interests. with higher altitudes to 1 500 masl. The transcription expression profile of neuropeptides that we report here and other characteristics such as the short peptide sequence (CCAP, nine amino acid residues, corazonin, References eleven amino acid residues and ETH, seventeen amino acid residues), the quaternary structure, the potential 1. Organización Mundial de la Salud. Enfermedades transmitidas por generation of peptidomimetics and modifications in vectores 2016. Ginebra: OMS 2016. Available at: http://www.who.int/ the amino acids residues to obtain synthetic structures mediacentre/factsheets/fs387/es/ with desired molecular properties (increased biostabil- 2. Tercero-Gutiérrez MJ, Olalla-Herbosa R. Enfermedades tropicales transmitidas por vectores. Medidas preventivas y profilaxis. Offarm ity, specificity, and permeability) provide advantages 2011;30:78-89. to these neuropeptides for further functional studies 3. Estevez-Lao TY, Boyce DS, Honegger HW, Hillyer JF. Cardioaccelera- in An. albimanus. tory function of the neurohormone CCAP in the mosquito Anopheles Orcokinin and ILP5 showed the highest transcrip- gambiae. J Exp Biol 2013;216:601-613. https://doi.org/10.1242/jeb.077164 tion levels in first instar larvae, while ILP2 showed the 4. Wanjala CL, Mbugi JP, Ototo E, Gesuge M, Afrane YA, Atieli HE, et al. highest transcription levels in pupae, allatostatin-A in Pyrethroid and DDT Resistance and Organophosphate Susceptibility adult mosquitoes and bursicon in third instar. On the among Anopheles spp. Mosquitoes, Western Kenya. Emerg Infect Dis 2015;21:2178-2181. https://doi.org/10.3201/eid2112.150814 other hand, allatostatin-A , bursicon and ILP2 showed 5. World Health Organization. World Malaria Report 2013. Geneva: WHO, lower transcription levels of in fourth instar larvae while 2013. Available at: http://www.who.int/malaria/publications/world_ma- orcokinin in adult mosquitoes and ILP5 in pupae. It laria_report_2013/en/ would be interesting to conduct further functional stud- 6. Dhadialla TS, Carlson GR, Le DP. New insecticides with ecdysteroidal ies and explore the possibility of blocking the expression and juvenile hormone activity. 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